a) Field of the Invention
The invention is directed to a method for characterizing a highly parallelized liquid handling technology using microplates and a test kit for carrying out the method. It is used wherever highly parallelized liquid handling technology is to be characterized with respect to accuracy and precision, particularly when the handled volumes lie within the μl range or sub-μl range and the characterization must be carried out under conditions corresponding to the real operating conditions of the liquid handling technology. Liquid handling technology using a large number of channels for sample handling, e.g., multipipettes with 96,384 or more channels, provides a large number of sample volumes in individual wells of a microplate that are arranged in grids for preparation and evaluation of samples in many channels or storage and transport, etc., thereof. The correctness and accuracy of sample handling is critically important for the quality and usability of the analysis results. For this reason, a check criterion which might be helpful as a measure of quality, as a basis for certification, and the like, becomes increasingly important for the supplier and for the user of such liquid handling technology
b) Description of the Related Art
In recent years, highly parallelized, extremely miniaturized methods of analysis based on microplate technology have led to the development of a large number of new and effective applications, particularly for target-oriented active ingredients for analysis of genomes and proteomes and for numerous other areas of biotechnology, medicine and environmental research. For the reasons described above, a corresponding highly parallelized technique for many-channel dispensing, reader technology and other technological developments capable of being adapted to the latter which could be characterized in a manner approaching application as closely as possible were created for many-channel handling of samples. Further, the characterization of a large number of individual channels should be practicable, must not require excessive expenditure on analysis and should be sufficiently precise.
As has been well known for a long time with respect to a large quantity of pipettes, gravimetric methods are used for this characterization along with methods which measure the dilution of an analytic signal of a sample by a diluent, this analytic signal being, in itself, easy to track. Examples of signals of this type are optical signals or radioactivity of a sample.
Gravimetric methods are very precise, but are hardly usable for the μl range and sub-μl range insofar as assessments of precision and accuracy are required to fall within a range of better than 0.5%. Further, the use of gravimetric methods is rendered nearly impossible firstly by evaporation, which constitutes a severe hindrance especially within this volume range, and secondly by problems relating to practicability (very many individual channels must now be characterized, whereas previously only 1 to a maximum of 12 channels had to be characterized). Examinations of this kind were previously restricted to relatively large individual volumes (e.g., GIT Laborzeitschrift 11/2001, 1185-86). The realization of conditions approximating those of real application also creates problems for characterization.
Photometric methods for calibrating pipettes were already described in the 1980s. For example, U.S. Pat. No. 4,354,376 describes a kit for calibrating pipettes which is based on the principle of measuring the dilution of dye solutions. U.S. Pat. No. 5,492,673 describes a reagent system for colorimetric calibration of pipettes which uses a special mixture of substances to correct for the path length of the round cells or cuvettes that are used in order to prevent nonlinearities in the measured absorbances as a function of dye concentration brought about by agglomeration and to improve the stability of the proposed reagent kit. The mixture comprises a 2-buffer system, each with a color indicator. These color indicators differ sharply in the position of the wavelengths of the maxima of the light absorption. Further, the mixture contains substances which inhibit aglomerization and improve stability. However, considerable expenditure on correction is required to exclude device-specific influences due to the photometer that is used and due to the cuvettes and influences particularly of the surrounding temperature.
The availability of parallel reader technology invites the characterization of parallelized dispensing technique using this technology, especially since the large number of individual channels to be characterized would otherwise be very difficult to calibrate. However, it must be taken into consideration that reader technology for microplates is based on the principle of vertical photometry; that is, it has no fixed path length for the individual well. The path length is determined by the volume that is used and by the developing meniscus and is subject to considerable variability depending upon the surface characteristics of the analyte and the mechanical conditions under which the microplate is handled. In U.S. Pat. No. 6,188,476, the absorbance of water in the infrared range is made use of to normalize the measured absorbances of the analyte with respect to a uniform path length in order to compensate for uncertainty regarding the path length. However, practical experience shows that although the average path length can be determined by this correction, compensation of the influence of the individual menisci is unsatisfactory.
In the simplest case, when an absorbance-measuring reader is used to measure the sample volume of a channel of the liquid handling technology with n channels, a diluent volume VD is introduced in the individual wells of the microplate and a sample volume VP which contains a dye F1 in a concentration CPF1 and which is to be determined is added thereto and mixed. The measured n absorbances of the sample solution AP and the n mixtures AM are functions of the respective concentrations of solutions and the path length d according to the Beer-Lambert law:AP=εF1*CPF1*dP AM=εF1*CMF1*dM,where CMF1 is the concentration of dye F1 in the mixture and ε designates the extinction coefficient of the indicator.
The dilution factor DF DF=CMF1/CPF1=VP/(VP+VD)can be used to determine VP.VP=VD*DF/(1−DF)=VD*CMF1/CPF1/(1−CMF1/CPF1)  (1)
It can be seen from equation (1) that the precision with which VP is determined depends upon the precision with which two absorbances are determined in the reader and upon the accuracy of the present volume VD.
The accuracy of the absorbances A measured in the reader depends upon multiplicatively acting (f) and additively acting (a) errors:A=A*f+a. 
Multiplicatively acting errors are chiefly the path length which varies because of meniscus formation and the temperature-dependent changes in ε; additively acting errors are brought about, for example, by the formation of bubbles, which is frequently observed, and by deposits, scratches and fizz or lint which are sometimes observed. These errors, which ultimately influence the analytic results of the volume determination, can be eliminated in large part by multiwavelength photometry. A procedure of this kind is described, for example, for determining temperature in microplates with thermochrome indicators by absorbance measurement (DE 199 28 056). Practical investigations of the variability of the absorbances measured in readers show that while good precision of the relative values of the absorbances in the individual wells of a microplate with respect to the mean of all measured wells (intra-assay precision) is achieved through the use of multiwavelength photometry, the individual values and mean values of the absorbances measured and obtained, respectively, for different plates have unacceptably high deviations.
Further, with regard to the use of reader technology for characterizing multipipettes, it must be taken into consideration that there are presently no readers for microplates with well densities greater than 384 per microplate (particularly 1536 or more wells) which can measure light absorbances with sufficient accuracy.